Methods and pharmaceutical compositions for the treatment of   cardiomyopathy due to friedreich ataxia

ABSTRACT

A method for preventing or treating cardiomyopathy due to energy failure in a subject in need thereof is provided. The method comprises administering to the subject a therapeutically effective amount of a vector which comprises a nucleic acid sequence encoding a gene that can reverse energy failure. An exemplary cardiomyopathy is that which is associated with Friedreich ataxia and an exemplary nucleic acid sequence comprises a nucleic acid that encodes frataxin (FXN).

FIELD OF THE INVENTION

The present invention relates a method for preventing or treatingcardiomyopathy due to energy failure in a subject in need thereof,comprising administering to said subject a therapeutically effectiveamount of a vector which comprises a nucleic acid sequence of a genethat can reverse energy failure.

More particularly, the invention relates to a method for preventing ortreating a cardiomyopathy associated with Friedreich ataxia in a subjectin need thereof, comprising administering to said subject atherapeutically effective amount of a vector which comprises a frataxin(FXN) encoding nucleic acid.

BACKGROUND OF THE INVENTION

Friedreich ataxia (FRDA), an autosomal progressive recessiveneurodegenerative disorder associated with cardiomyopathy, is caused byreduced expression of the mitochondrial protein, frataxin [V. Campuzanoet al., 1996 and V. Campuzano et al., 1997]. The cardiomopathyassociated with FRDA is hypertrophic. As the disease progresses, thereis a natural transition from hypertrophy to dilation, with death ofcardiomyocytes replaced by fibrotic tissue leading to systolic anddiastolic dysfunction [R. M. Payne et al., 2012]. Impaired myocardialperfusion reserve index associated with microvascular dysfunction andfibrosis occurs even prior to the onset of overt cardiomyopathy.Consistent with impaired mitochondrial respiratory chain function thatleads to energy deficit, phosphorus magnetic resonance spectroscopyshows reduced ATP production in patient heart, which strongly correlateswith the degree of cardiac hypertrophy. Cardiac dysfunction,predisposing to congestive heart failure and supraventriculararrhythmias, is the primary mode of death in ˜60% of patients with FRDA.

Although the function of frataxin is still under investigation,available evidence supports a role as an activator of iron-sulfur (Fe—S)cluster biogenesis in mitochondria [C. L. Tsai et al., 2010 andSchmucker et al., 2012]. In particular, frataxin was recently shown tocontrol iron delivery for de novo Fe—S cluster biogenesis throughactivation of cysteine desulfurase activity [Colin et al., 2013].

Fe—S clusters are essential prosthetic groups for many proteins with avariety of functions and subcellular localizations. Frataxin deficiencyleads to impairment of Fe—S cluster-containing proteins, alteredcellular iron metabolism, mitochondrial dysfunction and increasedsensitivity to oxidative stress. Most mitochondrial and biochemicaldefects identified in human patients have been recapitulated in mousemodels of FRDA [H. Puccio et al., 2001 and Simon et al. et al., 2004],providing valuable models for testing potential therapeuticinterventions. Particularly, the MCK conditional mouse model, withcomplete frataxin deletion in cardiac and skeletal muscle, recapitulatesthe cardiomyopathy observed in FRDA patients with a more rapidlyprogressive course [H. Puccio et al., 2001 and H. Seznec et al., 2004].Furthermore, the MCK mouse demonstrated that hypertrophy andmitochondrial Fe—S cluster protein defects precede mitochondrial ironaccumulation and increase sensitivity to oxidative stress.

To date, no treatment exists for stopping or slowing down thecardiomyopathy of FRDA. Current therapeutic approaches in clinical useor under evaluation are directed at alleviating symptoms and maximizingquality of life [R. B. Wilson 2012]. Thus, there is an important needfor a novel therapeutic approach to treat cardiomyopathy associated withFriedreich ataxia.

SUMMARY OF THE INVENTION

In the present invention, the therapeutic effect of an AVVrh10 vectorcarrying a human frataxin cDNA on the cardiac phenotype in a mammalianmodel of FRDA cardiomyopathy was investigated. The results showed thatdelivery of a vector encoding hFXN resulted in i) prevention of thedevelopment of disease symptoms in asymptomatic individuals and ii)reversal of disease symptoms in individuals who already exhibitedcardiomyopathy, mitochondrial dysfunction and the biochemical impairmentassociated with frataxin deficiency.

More generally, the inventors show that by restoring an essentialmitochondrial function with the use of the nuclear-encoded FXN gene, andthereby increasing the energy production of the mitochondria, themyocardium function can be restored and the interstitial cardiacfibrosis stopped. Considering that inefficient energy utilization andincreased energy demand by the sarcomere have been suggested as a keyconsequence of many, if not all, hypertrophic cardiomyopathy associatedmutations (Sweeney H L et al., 1998), the results demonstrate that theuse of a gene that can reverse energy failure is useful for preventingor treating cardiomyopathy linked to or associated with energy failure.

Thus, the invention relates to a method for preventing or treatingcardiomyopathy due to energy failure in a subject in need thereof,comprising administering to said subject a therapeutically effectiveamount of a vector which comprises a nucleic acid sequence of a genethat can reverse energy failure.

More particularly, the invention relates to a method for preventing ortreating a cardiomyopathy associated with Friedreich ataxia in a subjectin need thereof, comprising administering to said subject atherapeutically effective amount of a vector which comprises a frataxin(FXN) encoding nucleic acid.

DETAILED DESCRIPTION OF THE INVENTION Methods of the Invention

A first object of the invention relates to a method for preventing ortreating cardiomyopathy due to energy failure in a subject in needthereof, comprising administering to said subject a therapeuticallyeffective amount of a vector which comprises a nucleic acid sequence ofa gene that can reverse energy failure.

By “energy failure” we mean inadequate or abnormal energy production bymitochondria, and/or lower than normal levels of ATP production. Thephrase “reverse energy failure” denotes that energy failure is reversedand/or that energy metabolism is returned or restored to a normal,non-pathological state, or is at least improved compared to the state orlevel that would be present if a treatment were not administered. Forexample, a “reversal of energy failure” may involve an increase in orrestoration of mitochondrial function as a result of providing a patientwith a gene that can reverse energy failure such as the exemplary FXNgene. In one embodiment, the invention relates to a method forpreventing or treating cardiomyopathy due to energy failure in a subjectin need thereof, comprising administering to the subject atherapeutically effective amount of a vector which comprises a nucleicacid sequence of a gene that can reverse the pathological effects ofenergy function, e.g. cardiomyopathy.

As used herein the term “cardiomyopathy due to energy failure” denotesone or more of a deterioration of the function of the myocardium leadingto heart failure, cardiac remodelling, long-term high blood pressure,heart valve problems, impaired calcium cycling sensitivity, disturbedbiochemical stress sensing, altered energy homeostasis due but notlimited to mitochondrial dysfunction and fibrosis.

In a particular embodiment, the cardiomyopathy due to energy failure maybe one or more of a dilated cardiomyopathy, a hypertrophiccardiomyopathy, a restrictive cardiomyopathy or an ischemiccardiomyopathy.

In another particular embodiment, the cardiomyopathy due to energyfailure may be a cardiomyopathy due to a deficiency of fatty oxidation,including but not limited to primary carnitine deficiency, long-chain3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency, translocasedeficiency, and very long-chain acyl-CoA dehydrogenase (VLCAD)deficiency.

In another particular embodiment, the cardiomyopathy due to energyfailure may be a cardiomyopathy associated with Friedreich ataxia.

As used herein, the term “a gene that can reverse energy failure”denotes a nuclear or mitochondrial gene that can reverse energy failureand/or mitochondrial dysfunction.

In a particular embodiment, the gene that can reverse energy failure maybe a nuclear gene encoding a subunit of pyruvate dehydrogenase complex,a nuclear or a mitochondrial gene coding for a subunit of Complex I,III, IV or V involved in oxidative phosphorylation; a mitochondrial geneencoding transfer RNA, a gene involved in the biogenesis of mitochondriasuch as SIRT1, a gene involved in the fusion of mitochondria such asOPA1, a gene involved in the fission of mitochondria such as FIS1 or agene involved in the oxidation of fatty acid such as long-chain3-hydroxyacyl-CoA dehydrogenase or very long-chain specific acyl-CoAdehydrogenase.

In a particular embodiment, the gene that can reverse energy failure isthe frataxin (FXN) gene.

As used herein in its broadest meaning, the term “preventing” or“prevention” refers to preventing the disease or condition fromoccurring in a subject which has not yet been diagnosed as having it.The subject may, however, be known to be susceptible to developing thedisease, e.g. may be known or suspected of harbouring a genetic mutationthat may lead to the condition, even though overt clinical symptoms havenot yet appeared.

As used herein, the term “treating” or “treatment” means reversing,alleviating, or inhibiting the progress of the disorder or condition towhich such term applies, or one or more symptoms of such disorder orcondition. A “therapeutically effective amount” is intended for aminimal amount of active agent which is necessary to impart therapeuticbenefit to a subject. For example, a “therapeutically effective amount”to a patient is such an amount which induces, ameliorates, stabilises,slows down the progression or otherwise causes an improvement in thepathological symptoms, disease progression or physiological conditionsassociated with or resistance to succumbing to a disorder.

As used herein, the term “gene” refers to a polynucleotide containing atleast one open reading frame that is capable of encoding a particularpolypeptide or protein after being transcribed and translated.

As used herein, the terms “coding sequence”, “a sequence which encodes aparticular protein” or “encoding nucleic acid”, denotes a nucleic acidsequence which is transcribed (in the case of DNA) and translated (inthe case of mRNA) into a polypeptide in vitro or in vivo when placedunder the control of (operably linked to) appropriate regulatorysequences. The boundaries of the coding sequence are determined by astart codon at the 5′ (amino) terminus and a translation stop codon atthe 3′ (carboxy) terminus. A coding sequence can include, but is notlimited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNAsequences from prokaryotic or eukaryotic DNA, and even synthetic DNAsequences.

In a particular embodiment, the invention relates to a method forpreventing or treating a cardiomyopathy associated with Friedreichataxia in a subject in need thereof, comprising administering to saidsubject of a therapeutically effective amount of a vector whichcomprises a frataxin (FXN) encoding nucleic acid.

The FXN gene encodes the protein frataxin. Frataxin is a proteinlocalized to the mitochondrion and is involved in assembly ofiron-sulfur clusters by regulating iron entry and the activity ofcysteine desulfurase. A cDNA sequence for human FXN (transcriptvariant 1) is disclosed in Genbank Access Number NM_(—)000144 orNG_(—)008845 (SEQ ID NO:1). The amino acid sequence of human frataxin isshown in SEQ ID NO:2.

The sequence of the nucleic acid of the frataxin (cDNA) is:

(SEQ ID NO: 1)agtctccctt gggtcagggg tcctggttgc actccgtgct ttgcacaaag caggctctcc attttgtta aatgcacgaatagtgctaag ctgggaagtt cttcctgagg tctaacctct agctgctccc ccacagaaga gtgcctgcgg ccagtggccaccaggggtcg ccgcagcacc cagcgctgga gggcggagcg ggcggcagac ccggagcagc atgtggactctcgggcgccg cgcagtagcc ggcctcctgg cgtcacccag cccagcccag gcccagaccc tcacccgggt cccgcggccggcagagttgg ccccactctg cggccgccgt ggcctgcgca ccgacatcga tgcgacctgc acgccccgcc gcgcaagttcgaaccaacgt ggcctcaacc agatttggaa tgtcaaaaag cagagtgtct atttgatgaa tttgaggaaa tctggaactttgggccaccc aggctctcta gatgagacca cctatgaaag actagcagag gaaacgctgg actctttagc agagttttttgaagaccttg cagacaagcc atacacgttt gaggactatg atgtctcctt tgggagtggt gtcttaactg tcaaactgggtggagatcta ggaacctatg tgatcaacaa gcagacgcca aacaagcaaa tctggctatc ttctccatcc agtggacctaagcgttatga ctggactggg aaaaactggg tgtactccca cgacggcgtg tccctccatg agctgctggc cgcagagctcactaaagcct taaaaaccaa actggacttg tcttccttgg cctattccgg aaaagatgct tgatgcccag ccccgttttaaggacattaa aagctatcag gccaagaccc cagcttcatt atgcagctga ggtctgtttt ttgttgttgt tgttgtttat tttttttattcctgcttttg aggacagttg ggctatgtgt cacagctctg tagaaagaat gtgttgcctc ctaccttgcc cccaagttctgatttttaat ttctatggaa gattttttgg attgtcggat ttcctccctc acatgatacc ccttatcttt tataatgtct tatgcctatacctgaatata acaaccttta aaaaagcaaa ataataagaa ggaaaaattc caggagggaa aatgaattgt cttcactcttcattctttga aggatttact gcaagaagta catgaagagc agctggtcaa cctgctcact gttctatctc caaatgagacacattaaagg gtagcctaca aatgttttca ggcttctttc aaagtgtaag cacttctgag ctctttagca ttgaagtgtcgaaagcaact cacacgggaa gatcatttct tatttgtgct ctgtgactgc caaggtgtgg cctgcactgg gttgtccagggagacctagt gctgtttctc ccacatattc acatacgtgt ctgtgtgtat atatattttt tcaatttaaa ggttagtatg gaatcagctgctacaagaat gcaaaaaat ttccaaagac aagaaaagag gaaaaaaagc cgttttcatg agctgagtga tgtagcgtaacaaacaaaat catggagctg aggaggtgcc ttgtaaacat gaaggggcag ataaaggaag gagatactca tgttgataaagagagccctg gtcctagaca tagttcagcc acaaagtagt tgtccctttg tggacaagtt tcccaaattc cctggacctctgcttcccca tctgttaaat gagagaatag agtatggttg attcccagca ttcagtggtc ctgtcaagca acctaacagctagttctaa ttccctattg ggtagatgag gggatgacaa agaacagttt ttaagctata taggaaacat tgttattggtgttgccctat cgtgatttca gttgaattca tgtgaaaata atagccatcc ttggcctggc gcggtggctc acacctgtaatcccagcact tttggaggcc aaggtgggtg gatcacctga ggtcaggagt tcaagaccag cctggccaac atgatgaaacccgtctcta ctaaaaatac aaaaaattag ccgggcatga tggcaggtgc ctgtaatccc agctacttgg gaggctgaagcggaagaatc gcttgaaccc agaggtggag gttgcagtga gccgagatcg tgccattgca ctgtaacctg ggtgactgagcaaaactctg tctcaaaata ataataacaa tataataata ataatagcca tcctttattg tacccttact gggttaatcg tattataccacattacctca ttttaatttt tactgacctg cactttatac aaagcaacaa gcctccagga cattaaaatt catgcaaagt tatgctcatgttatattatt ttcttactta aagaaggatt tattagtggc tgggcatggt ggcgtgcacc tgtaatccca ggtactcaggaggctgagac gggagaattg cttgacccca ggcggaggag gttacagtga gtcgagatcg tacctgagcg acagagcgagactccgtctc aaaaaaaaaa aaaaggaggg tttattaatg agaagtttgt attaatatgt agcaaaggct tttccaatgggtgaataaaa acacattcca ttaagtcaag ctgggagcag tggcatatac ctatagtccc agctgcacag gaggctgagacaggaggatt gcttgaagcc aggaattgga gatcagcctg ggcaacacag caagatccta tctcttaaaa aaagaaaaaaaaacctatta ataataaaac agtataaaca aaagctaaat aggtaaaata ttttttctga aataaaatta ttttttgagt ctgatggaaatgtttaagtg cagtaggcca gtgccagtga gaaaataaat aacatcatac atgtttgtat gtgtttgcat cttgcttcta ctgaaagtttcagtgcaccc cacttactta gaactcggtg acatgatgta ctcctttatc tgggacacag cacaaaagag gtatgcagtggggctgctct gacatgaaag tggaagttaa ggaatctggg ctcttatggg gtccttgtgg gccagccctt caggcctattttactttcat tttacatata gctctaattg gtttgattat ctcgttccca aggcagtggg agatccccat ttaaggaaagaaaaggggcc tggcacagtg gctcatgcct gtaatcccag cactttggga ggctgaggca agtgtatcac ctgaggtcaggagttcaaga ccagcctggc caacatggca aaatcccgtc tctactaaaa atattaaaaa attggctggg cgtggtggttcgtgcctata atttcagcta ctcaggaggc tgaggcagga gaatcgctgt aacctggggg gtggaggttg cagtgagacgagatcatgcc acttcactcc agcctggcca acagagcca actccgtctc aaataaataa ataaataaat aaagggacttcaaacacatg aacagcagcc aggggaagaa tcaaaatcat attctgtcaa gcaaactgga aaagtaccac tgtgtgtaccaatagcctcc ccaccacaga ccctgggagc atcgcctcat ttatggtgtg gtccagtcat ccatgtgaag gatgagtttccaggaaaagg ttattaaata ttcactgtaa catactggag gaggtgagga attgcataat acaatcttag aaaacttttttttccccttt ctattttttg agacaggatc tcactttggc actcaggctg gaggacagtg gtacaatcaa agctcatggcagcctcgacc tccctgggct tgggcaatcc tcccacaggt gtgcacctcc atagctggct aatttgtgta ttttttgtagagatggggtt tcaccatgtt gcccaggctg gtctctaaca cttaggctca agtgatccac ctgcctcgtc ctcccaagatgctgggatta caggtgtgtg ccacaggtgt tcatcagaaa gctttttcta ttatttttac cttcttgagt gggtagaacctcagccacat agaaaataaa atgttctggc atgacttatt tagctctctg gaattacaaa gaaggaatga ggtgtgtaaaagagaacctg ggtttttgaa tcacaaattt agaatttaat cgaaactctg cctcttactt gtttgtagac actgacagtg gcctcatgtttttttttttt ttaatctata aaatggagat atctaacatg ttgagcctgg gcccacaggc aaagcacaat cctgatgtgagaagtactca gttcatgaca actgttgttc tcacatgcat agcataattt catattcaca ttggaggact tctcccaaaatatggatgac gttccctact caaccttgaa cttaatcaaa atactcagtt tacttaactt cgtattagat tctgattccc tggaaccatttatcgtgtgc cttaccatgc ttatatttta cttgatcttt tgcatacctt ctaaaactat tttagccaat ttaaaatttg acagtttgcattaaattata ggtttacaat atgctttatc cagctatacc tgccccaaat tctgacagat gcttttgcca cctctaaaggaagacccatg ttcatagtga tggagtttgt gtggactaac catgcaaggt tgccaaggaa aaatcgcttt acgcttccaaggtacacact aagatgaaag taattttagt ccgtgtccag ttggattctt ggcacatagt tatcttctgc tagaacaaactaaaacagct acatgccagc aagggagaaa ggggaaggag gggcaaagtt ttgaaatttc atgtaaattt atgctgttcaaaacgacgag ttcatgactt tgtgtataga gtaagaaatg ccttttcttt tttgagacag agtcttgctc tgtcacccaggctggagtgc agtggcacga tctgggctca ctacaacctc cgcctcctgg gttcaagcaa ttctctgcct cagcctcccgagtagctggg attacaggtg cctgccacca cacccggcta atttttgtat ttttagtaga gacggggttt caccatcatggccaggctgg tcttgaactc ctgacctagt aatccacctg cctccgcctc ccaaagtgct gggattacag gcgtgagccactgcacccag ccagaaatgc cttctaatct ttggtttatc ttaattagcc aggacacttg gagtgcatcc cgaagtacctgatcagtggc ccctttggaa tgtgtaaaac tcagctcact tatatccctg catccgctac agagacagaa tccaagctcatatgttccat cttctctggc tgtatagttt aaggaatgga aggcaccaga acagatttat tgaaatgttt attagctgaa gatttatttagacagttgag gaaaacatca gcacccagca gtaaaattgg ctctcaaaga ttttcttctc ctgtggaaag tcagacctctgaggccccat ccaggtagaa gtactagtgc aagaagggcc tctgctgtcc acttgtgttt ctgtgatctg tgggaacattgttaacgcca catcttgacc tcaaattgtt tagctcctgg ccagacacgg tggctcacac ctgtaatccc agcactttgagaggctgagg caggtggatc acctgaggtt aggagttcga ggccagcctg gtcaacatgg taaaaccccg cctctactaaaaatacaaaa attagctggc cgtagtggcg cacgcctgtt atcccagcta ctcgggaggc tgaggcagga gaattgcttgaacctgggtg gtggaggttg cagtgagccg agattacacc actgcactcc agcctgggtg acaagaggga aactccattaaaaaaatgta attcccgtgt ctgccatctt aagtgtaaag gtggctaaat tatatagaaa aataagacaa tatcatttcccaattacatt cctttcctac cgcactctat gatgctagct gagatttttc caaaagaaaa tggcttaaat aaaaccctagagaaagaaa aactttaaat ccctccaaag ctcaaaagta atagaaacag atgagtttgg agtcaggatt tctctgtaagattgcctagg ctgtgtactg cacatctcca ggtgccactg ttgacagaga ttataactac aatgtgaagt gaatggtgccactgacagtt atgcaaaccg tccagagcat agccacctga tcctgctggg attcctcttg ccagtccatc agcagttccccttgaaagtt tcaccaaaca tcccttaaat ctgccctctc ctgcccgtcc ccagtggagg tcctcatcat ttttcacctgcatttttgca ggagctttct tatatccacc ttcctccttt tctctcagcc catcatctag ctacacagtc tccagggtaa gctttcagaaaggcaatctc ttgtctgtaa aacctaagca ggaccaaggc caagtttctt agcctgaaaa atgtgctttt ctgactgaactgttcaggca ctgactctac atataattat gcttttctac cccctcacac tcaacacttt gactccagca atcccaaatccccagatccc taagtgtgct gtgctatttt cacgtggctc tcagacttgg ccagtgctgt ttccattttg gtctttattc cccacatctctgcctggggg gtagattcta ccctgaaaaa tgttcttggc acagccttgc aaactcctcc tccactcagc ctctgcctggatgcccttga ttgttccatg tcctcagcat accatgtttg tctttcccag cactgaccta ccatgtgtca cccctgcttg gctgtaccttccatgaggct aggactatgt gtctcctttg ttgactgctg ttgccctagc atcttgcaca gttccttgca cacaattagagctctataaa tgtcaaataa atgtgttata attatatgtt taagatagtt gttcaaataa actctaaata accccaac. 

The sequence of the frataxin protein is (SEQ ID NO:2):

MWTLGRRAVAGLLASPSPAQAQTLTRVPRPAELAPLCGRRGLRTDIDATCTPRRASSNQRGLNQIWNVKKQSVYLMNLRKSGTLGHPGSLDETTYERLAEETLDSLAEFFEDLADKPYTFEDYDVSFGSGVLTVKLGGDLGTYVINKQTPNKQIWLSSPSSGPKRYDWTGKNWVYSHDGVSLHELLAAELTKALKTKLDL SSLAYSGKDA.

In a particular embodiment, the invention provides a nucleic acidconstruct comprising a sequence that is or includes SEQ ID NO:1 or avariant thereof for treating cardiomyopathy associated with Friedreichataxia.

The variants include, for instance, naturally-occurring variants due toallelic variations between individuals (e.g., polymorphisms),alternative splicing forms, in particular transcript variants 2 and 3(GenBank accession numbers NM_(—)001161706 and NM_(—)181425), etc. Theterm variant also includes FXN gene sequences from other sources ororganisms. Variants are preferably substantially homologous to SEQ IDNO:1, i.e., exhibit a nucleotide sequence identity of typically at leastabout 75%, preferably at least about 85%, more preferably at least about90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% with SEQID NO: 1. Variants of a FXN gene also include nucleic acid sequences,which hybridize to a sequence as defined above (or a complementarystrand thereof) under stringent hybridization conditions. Typicalstringent hybridisation conditions include temperatures above 30° C.,preferably above 35° C., more preferably in excess of 42° C., and/orsalinity of less than about 500 mM, preferably less than 200 mM.Hybridization conditions may be adjusted by the skilled person bymodifying the temperature, salinity and/or the concentration of otherreagents such as SDS, SSC, etc.

In a particular embodiment, the variant may be a variant of the SEQ IDNO:1 which encodes for the amino acid sequence 81-210 of the SEQ ID NO:2(named variant “81-210”).

In a another particular embodiment, a sequence known as the“mitochondrion-targeting signal” or “mitochondrial targeting signal” maybe added to a variant of the invention, for example to the variant“81-210”. Sequences known as “mitochondrion-targeting signal” or“mitochondrial targeting signal” are referred to as MTS by the skilledperson.

A MTS sequence can be identified within a protein or nucleic acidsequence by a person of ordinary skill in the art.

Most mitochondrion-targeting peptides consist of an N-terminalpre-sequence of about 15 to 100 residues, preferably of about 20 to 80residues. They are enriched in arginine, leucine, serine and alanine.Mitochondrial pre-sequences show a statistical bias of positivelycharged amino acid residues, provided mostly through arginine residues;very few sequences contain negatively charged amino acids.Mitochondrion-targeting peptides also share an ability to form anamphilic alpha-helix.

A complete description of a method to identify a MTS is available in: M.G. Claros, P. Vincens, 1996 (Eur. J. Biochem. 241, 779-786 (1996),“Computational method to predict mitochondrially imported proteins andtheir targeting sequences”), the complete content of which is hereinincorporated by reference.

In another embodiment, the invention relates to a method for treating orpreventing diseases associated with frataxin deficiency in a subject inneed therefore, comprising administering to said subject atherapeutically effective amount of a vector which comprises a nucleicacid encoding frataxin.

In another embodiment, the invention relates a method for preventing ortreating cardiomyopathy due but not limited to a cause such as energyfailure in a subject in need thereof, comprising administering to saidsubject a therapeutically effective amount of a vector which comprises anucleic acid sequence of a gene that can reverse energy failure.

In another particular embodiment, the invention relates a method forpreventing or treating cardiomyopathy due but not limited to a causesuch as energy failure in a subject in need thereof, comprisingadministering to said subject a therapeutically effective amount of avector which comprises a frataxin (FXN) encoding nucleic acid.

Non Viral Vectors

In a particular embodiment, the vector use according to the invention isa non viral vector. Typically, the non viral vector may be a plasmidwhich includes nucleic acid sequences encoding FXN gene, or variantsthereof, as described above.

The Viral Vectors

Gene delivery viral vectors useful in the practice of the presentinvention can be constructed utilizing methodologies well known in theart of molecular biology. Typically, viral vectors carrying transgenesare assembled from polynucleotides encoding the transgene, suitableregulatory elements and elements necessary for production of viralproteins which mediate cell transduction.

The terms “gene transfer” or “gene delivery” refer to methods or systemsfor reliably inserting foreign DNA into host cells. Such methods canresult in transient expression of non-integrated transferred DNA,extrachromosomal replication and expression of transferred replicons(e.g. episomes), or integration of transferred genetic material into thegenomic DNA of host cells.

Examples of viral vector include but are not limited to adenoviral,retroviral, lentiviral, herpesvirus and adeno-associated virus (AAV)vectors.

Such recombinant viruses may be produced by techniques known in the art,such as by transfecting packaging cells or by transient transfectionwith helper plasmids or viruses. Typical examples of virus packagingcells include but are not limited to PA317 cells, PsiCRIP cells, GPenv+cells, 293 cells, etc. Detailed protocols for producing suchreplication-defective recombinant viruses may be found for instance inWO95/14785, WO96/22378, U.S. Pat. No. 5,882,877, U.S. Pat. No.6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056 andWO94/19478, the complete contents of each of which is herebyincorporated by reference.

In one embodiment, adeno-associated viral (AAV) vectors are employed.

In other embodiments, the AAV vector is AAV1, AAV2, AAV3, AAV4, AA5,AAV6, AAV7, AAV8, AAV9, AAVrh10 or any other serotypes of AAV that caninfect humans, monkeys or other species.

In an exemplary embodiment, the AAV vector is an AAVrh10.

By an “AAV vector” is meant a vector derived from an adeno-associatedvirus serotype, including without limitation, AAV-1, AAV-2, AAV-3,AAV-4, AAV-5, AAV6, etc. AAV vectors can have one or more of the AAVwild-type genes deleted in whole or part, preferably the rep and/or capgenes, but retain functional flanking inverted terminal repeat (ITR)sequences. Functional ITR sequences are necessary for the rescue,replication and packaging of the AAV virion. Thus, an AAV vector isdefined herein to include at least those sequences required in cis forreplication and packaging (e.g., functional ITRs) of the virus. The ITRsneed not be the wild-type nucleotide sequences, and may be altered, e.gby the insertion, deletion or substitution of nucleotides, as long asthe sequences provide for functional rescue, replication and packaging.AAV expression vectors are constructed using known techniques to atleast provide as operatively linked components in the direction oftranscription, control elements including a transcriptional initiationregion, the DNA of interest (i.e. the FXN gene) and a transcriptionaltermination region.

The control elements are selected to be functional in a mammalian cell.The resulting construct which contains the operatively linked componentsis bounded (5′ and 3′) with functional AAV ITR sequences. By“adeno-associated virus inverted terminal repeats” or “AAVITRs” is meantthe art-recognized regions found at each end of the AAV genome whichfunction together in cis as origins of DNA replication and as packagingsignals for the virus. AAV ITRs, together with the AAV rep codingregion, provide for the efficient excision and rescue from, andintegration of a nucleotide sequence interposed between two flankingITRs into a mammalian cell genome. The nucleotide sequences of AAV ITRregions are known. See, e.g., Kotin, 1994; Berns, K I “Parvoviridae andtheir Replication” in Fundamental Virology, 2nd Edition, (B. N. Fieldsand D. M. Knipe, eds., the complete contents of which is herebyincorporated by reference) for the AAV-2 sequence. As used herein, an“AAV ITR” does not necessarily comprise the wild-type nucleotidesequence, but may be altered, e.g., by the insertion, deletion orsubstitution of nucleotides. Additionally, the AAV ITR may be derivedfrom any of several AAV serotypes, including without limitation, AAV-1,AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, etc. Furthermore, 5′ and 3′ ITRswhich flank a selected nucleotide sequence in an AAV vector need notnecessarily be identical or derived from the same AAV serotype orisolate, so long as they function as intended, i.e., to allow forexcision and rescue of the sequence of interest from a host cell genomeor vector, and to allow integration of the heterologous sequence intothe recipient cell genome when AAV Rep gene products are present in thecell. Additionally, AAV ITRs may be derived from any of several AAVserotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV5, AAV-6, etc. Furthermore, 5′ and 3′ ITRs which flank a selectednucleotide sequence in an AAV expression vector need not necessarily beidentical or derived from the same AAV serotype or isolate, so long asthey function as intended, i.e., to allow for excision and rescue of thesequence of interest from a host cell genome or vector, and to allowintegration of the DNA molecule into the recipient cell genome when AAVRep gene products are present in the cell.

Particularly preferred are vectors derived from AAV serotypes havingtropism for and high transduction efficiencies in cells of the mammalianmyocardium, particularly cardiomyocytes and cardiomyocyte progenitors. Areview and comparison of transduction efficiencies of differentserotypes is provided in Cearley C N et al., Moleuclar Therapy 16(10);1710-1718, 2008, the complete contents of which is hereby incorporatedby reference. In other non-limiting examples, preferred vectors includevectors derived from any serotypes like AAV1, AAV2, AAV3, AAV4, AA5,AAV6, AAV7, AAV8, AAV9, or AAVrh10, which have also been shown totransduce cells of cardiomyocytes.

The selected nucleotide sequence is operably linked to control elementsthat direct the transcription or expression thereof in the subject invivo. Such control elements can comprise control sequences normallyassociated with the selected gene.

Alternatively, heterologous control sequences can be employed. Usefulheterologous control sequences generally include those derived fromsequences encoding mammalian or viral genes. Examples include, but arenot limited to, the phophoglycerate kinase (PKG) promoter, CAG (chickenbeta-actin promoter with CMV enhancer) promoter, MCK (muscle creatinekinase) promoter, the SV40 early promoter, mouse mammary tumor virus LTRpromoter; adenovirus major late promoter (Ad MLP); a herpes simplexvirus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMVimmediate early promoter region (CMVIE), rous sarcoma virus (RSV)promoter, synthetic promoters, hybrid promoters, and the like. Thepromoters can be of human origin or from other species, including frommice. In addition, sequences derived from nonviral genes, such as themurine metallothionein gene, will also find use herein. Such promotersequences are commercially available from, e.g. Stratagene (San Diego,Calif.).

Examples of heterologous promoters include but are not limited to theCMV promoter.

Examples of inducible promoters include but are not limited to DNAresponsive elements for ecdysone, tetracycline, and hypoxia andaufin.

The AAV expression vector which harbors the DNA molecule of interestbounded by AAV ITRs, can be constructed by directly inserting theselected sequence (s) into an AAV genome which has had the major AAVopen reading frames (“ORFs”) excised therefrom. Other portions of theAAV genome can also be deleted, so long as a sufficient portion of theITRs remain to allow for replication and packaging functions. Suchconstructs can be designed using techniques well known in the art. See,e.g. U.S. Pat. Nos. 5,173,414 and 5,139,941; International PublicationsNos. WO 92/01070 (published 23 Jan. 1992) and WO 93/03769 (published 4Mar. 1993); Lebkowski et al., 1988; Vincent et al., 1990; Carter, 1992;Muzyczka, 1992; Kotin, 1994; Shelling and Smith, 1994; and Zhou et al.,1994, the complete contents of each of which is hereby incorporated byreference. Alternatively, AAV ITRs can be excised from the viral genomeor from an AAV vector containing the same and fused 5′ and 3′ of aselected nucleic acid construct that is present in another vector usingstandard ligation techniques. AAV vectors which contain ITRs have beendescribed in, e.g. U.S. Pat. No. 5,139,941, the complete contents ofwhich is hereby incorporated by reference. In particular, several AAVvectors are described therein which are available from the American TypeCulture Collection (“ATCC”) under Accession Numbers 53222, 53223, 53224,53225 and 53226. Additionally, chimeric genes can be producedsynthetically to include AAV ITR sequences arranged 5′ and 3′ of one ormore selected nucleic acid sequences. Preferred codons for expression ofthe chimeric gene sequence in mammalian CNS cells can be used. Thecomplete chimeric sequence is assembled from overlappingoligonucleotides prepared by standard methods. See, e.g., Edge Nature,vol. 292, 1981, page 756; Nambair et al. Science, vol. 223, 1984, page1299; Jay et al. J. Biol. Chem. vol. 259, 1984, page 6311, the completecontents of each of which is hereby incorporated by reference. In orderto produce AAV virions, an AAV expression vector is introduced into asuitable host cell using known techniques, such as by transfection. Anumber of transfection techniques are generally known in the art. See,e.g. Graham et al, Virology, 52, 456-467, (1973); Sambrook et al. (1989)Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories,New York, Davis et al. (1986) Basic Methods in Molecular Biology,Elsevier, and Chu et al. (1981) Gene 13:197. Particularly suitabletransfection methods include calcium phosphate co-precipitation (Grahamet al., 1973), direct microinjection into cultured cells (Capecchi,1980), electroporation (Shigekawa et al., 1988), liposome mediated genetransfer (Mannino et al., 1988), lipid-mediated transduction (Feigner etal., 1987, PNAS USA, 84, 21, 7413-17), and nucleic acid delivery usinghigh-velocity microprojectiles (Klein et al., 1987, m Endocrinology120:2339-45). The complete contents of each of the foregoing referencesare hereby incorporated by reference in entirety.

For instance, a preferred viral vector, such as the AAVrh10, comprises,in addition to a FXN encoding nucleic acid sequence, the backbone of AAVvector with ITR derived from AAV-2, the promoter, such as the mouse PGK(phosphoglycerate kinase) gene or the cytomegalovirus/β-actin hybridpromoter (CAG) consisting of the enhancer from the cytomegalovirusimmediate gene, the promoter, splice donor and intron from the chickenβ-actin gene, the splice acceptor from rabbit β-globin, or any promotersuch as PGK, CAG, MCK.

Delivery of the Vectors

It is herein provided a method for treating cardiomyopathy due to energyfailure in a subject, said method comprising:

(a) providing a vector as defined above, which comprises a nucleic acidsequence of a gene that can reverse energy failure; and

(b) delivering the vector to the subject in need thereof and whereby thegene is expressed by the transduced cells at a therapeutically effectivelevel.

In a particular embodiment, a method for treating cardiomyopathyassociated with Friedreich ataxia in a subject is herein provided, saidmethod comprising:

(a) providing a vector as defined above, which comprises a FXN encodingnucleic acid; and

(b) delivering the vector to the subject in need thereof and whereby FXNis expressed by the transduced cells at a therapeutically effectivelevel.

In a particular method, the vector is delivered directly into themyocardium by epicardiac injection followed by minithoracotomy, byintracoronary injection, by endomyocardic injection or by another typeof injection useful in the heart.

Additional routes of administration may also comprise local applicationof the vector under direct visualization, e.g., superficial corticalapplication, or other nonstereotactic application. The vector may alsobe delivered, for example, intrathecally, into the ventricules or byintravenous injection.

The target cells of the vectors of the present invention are cells ofthe myocardium of a subject afflicted with a cardiomyopathy associatedwith Friedreich ataxia. Preferably the subject is a human being, adultor child. However, veterinary applications are also contemplated.

However the invention encompasses delivering the vector to biologicalmodels of the disease. In that case, the biological model may be anymammal at any stage of development at the time of delivery, e.g.,embryonic, fetal, infantile, juvenile or adult. Furthermore, the targetmyocardium cells may be essentially from any source, especially anycells derived from hiPS from FRDA patients, nonhuman primates andmammals of the orders Rodenta (mice, rats, rabbit, hamsters), Carnivora(cats, dogs), and Arteriodactyla (cows, pigs, sheep, goats, horses) aswell as any other non-human system (e.g. zebrafish model system).

The vectors used herein may be formulated in any suitable vehicle fordelivery. For instance they may be placed into a pharmaceuticallyacceptable suspension, solution or emulsion. Suitable mediums includesaline and liposomal preparations. More specifically, pharmaceuticallyacceptable carriers may include sterile aqueous of non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils such as oliveoil, and injectable organic esters such as ethyl oleate. Aqueouscarriers include but are not limited to water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like.

Preservatives and other additives may also be present such as, forexample, antimicrobials, antioxidants, chelating agents, and inert gasesand the like.

A colloidal dispersion system may also be used for targeted genedelivery. Colloidal dispersion systems include macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes.

The preferred doses and regimen may be determined by a physician, anddepend on the age, sex, weight, of the subject, and the stage of thedisease. As an example, for delivery of a nucleic acid sequence encodingan FXN polypeptide using a viral expression vector, each unit dosage ofFXN polypeptide expressing vector may comprise 2.5 to 100 μl of acomposition including a viral expression vector in a pharmaceuticallyacceptable fluid at a concentration ranging from 10¹¹ to 10¹⁶ viralgenome per ml, for example.

Pharmaceutical Composition

A second object of the invention concerns a pharmaceutical compositionfor preventing or treating cardiomyopathy due to energy failure in asubject in need thereof, which comprises a therapeutically effectiveamount of a vector which comprises a nucleic acid sequence of a genethat can reverse energy failure.

In a particular embodiment, the invention concerns a pharmaceuticalcomposition for preventing or treating cardiomyopathy associated withFriedreich ataxia in a subject in need thereof, which comprises atherapeutically effective amount of a vector which comprises a FXNencoding nucleic acid.

By a “therapeutically effective amount” is meant a sufficient amount ofthe vector of the invention to treat a cardiomyopathy associated withFriedreich ataxia at a reasonable benefit/risk ratio applicable to anymedical treatment.

It will be understood that the single dosage or the total daily dosageof the compounds and compositions of the present invention will bedecided by the attending physician within the scope of sound medicaljudgment. The specific therapeutically effective dose level for anyparticular patient will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; activity of thespecific compound employed; the specific composition employed, the age,body weight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific nucleic acid orpolypeptide employed; and like factors well known in the medical arts.For example, it is well within the skill of the art to start doses ofthe compound at levels lower than those required to achieve the desiredtherapeutic effect and to gradually increase the dosage until thedesired effect is achieved. However, the daily dosage of the productsmay be varied over a wide range per adult per day. The therapeuticallyeffective amount of the vector according to the invention that should beadministered, as well as the dosage for the treatment of a pathologicalcondition with the number of viral or non-viral particles and/orpharmaceutical compositions of the invention, will depend on numerousfactors, including the age and condition of the patient, the severity ofthe disturbance or disorder, the method and frequency of administrationand the particular peptide to be used.

The presentation of the pharmaceutical compositions that contain thevector according to the invention may be in any form that is suitablefor the selected mode of administration, for example, forintraventricular, intramyocardium, intracoronary or intravenousadministration.

In the pharmaceutical compositions of the present invention forintramuscular, intravenous, intramyocardium, intracoronary orintraventricular administration, the active principle, alone or incombination with one or more other active principles, can beadministered in a unit administration form, as a mixture withconventional pharmaceutical supports, to animals and human beings.

Preferably, the pharmaceutical compositions contain vehicles which arepharmaceutically acceptable for a formulation capable of being injected.These may be in particular isotonic, sterile, saline solutions(monosodium or disodium phosphate, sodium, potassium, calcium ormagnesium chloride and the like or mixtures of such salts), or dry,especially freeze-dried compositions which upon addition, depending onthe case, of sterilized water or physiological saline, permit theconstitution of injectable solutions.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid.It must be stable under the conditions of manufacture and storage andmust be preserved against the contaminating action of microorganisms,such as bacteria and fungi.

Solutions comprising compounds of the invention as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The vector according to the invention can be formulated into acomposition in a neutral or salt form. Pharmaceutically acceptable saltsinclude the acid addition salts (formed with the free amino groups ofthe protein) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed with thefree carboxyl groups can also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, histidine,procaine and the like.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetables oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminiummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activepolypeptides in the required amount in the appropriate solvent withseveral of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

Multiple doses can also be administered.

In another embodiment, the invention relates to a pharmaceuticalcomposition for treating or preventing diseases associated with frataxindeficiency in a subject in need therefore, comprising administering tosaid subject a therapeutically effective amount of a vector whichcomprises a nucleic acid encoding frataxin.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIG. 1 A, FIG. 1 B1, FIG. 1 B2. Administration of AAVrh10.CAG-hXN vectorat 3 weeks of age prevents the onset of cardiac failure and rescuessurvival in pre-symptomatic MCK mice. (FIG. 1 A) Survival rates ofwild-type (black solid line), treated (grey dotted line) and untreated(black dotted line) MCK mice. n=9-10 for each group. 100% survival wasobserved for wild type and treated mice up to 35 weeks and thus the twolines (i.e. the grey dotted line and the black dotted line) aresuperimposed near the top of the graph. (FIG. 1 B1 and FIG. 1 B2)Relative quantification of atrial natriuretic peptide (ANP), brainnatriuretic peptide (BNP) and sarcoplasmic reticulum Ca2+ ATPase(Serca2a) mRNA expressions in heart at 8 and 35 weeks for wild-type(white), treated (grey) and untreated (black) MCK mice; n=3-5 per group(*P<0.05; ***P<0.001). mRNA expression was normalized to 18S ribosomalRNA and is presented as a fold change relative to wild-type values. Dataare represented as means±SD.

FIG. 2 A1, FIG. 2 A2, FIG. 2 B1, FIG. 2 B2, FIG. 2 C, FIG. 2 D.Administration of AAVrh10.CAG-hXN vector at 7 weeks of age insymptomatic MCK mice with severe cardiac failure reverses the cardiaccontractile dysfunction, Fe—S cluster proteins, and cardiomyocyte andmitochondrial ultrastructure disorganization. (FIG. 2 A1 and FIG. 2 A2)Longitudinal echocardiographic assessment of the left ventricle mass(LVM, left) and the shortening fraction (SF, right) for wild-type (blackcircles) mice, treated (light grey squares) and untreated (greytriangle) MCK mice. Data are represented as means±SD. n=9-11 for eachgroup. (FIG. 2 B1 and FIG. 2 B2) Survival rates of wild-type mice (blacksolid line), treated (grey dotted line) and untreated (black dottedline) MCK mice. n=9-11 for each group. (FIG. 2 C) Relativequantification of atrial natriuretic peptide (ANP), brain natriureticpeptide (BNP) and sarcoplasmic reticulum Ca²⁺ ATPase (Serca2a) mRNAexpressions in heart at 8, 15 and 22 weeks for wild-type (white),treated (grey) and untreated (black) MCK mice; n=3-5 per group and perage (*P<0.05; **P<0.01; ns: statistically non-significant). mRNAexpression was normalized to 18S ribosomal RNA and is presented as afold change relative to wild-type values. Data are represented asmeans±SD. (FIG. 2 D) Biochemical measurements of combined cytosolic andmitochondrial aconitases (Aco) and succinate dehydrogenase (SDH, complexII) activities in heart from wild-type (white) mice, treated (grey) anduntreated (black) MCK mice at 8, 15 and 22 weeks of age; n=3-6 per groupand per age (**P<0.01). Isocitrate dehydrogenase (IDH) activity was usedto normalize SDH and aconitase activities for total mitochondrialcontent.

EXAMPLES Material & Methods

Adeno-Associated Viral Vector Construction and Production

Human frataxin (hFXN) cDNA, including the mitochondrial targetingsequence, fused to a HA tag was subcloned in a pAAV2-CAG plasmid(Sondhi, Hackett et al. 2007) to produce pAAV2-CAG-hFXN that includedthe viral inverted terminal repeat (ITR) from AAV2; thecytomegalovirus/β-actin hybrid promoter, consisting of the enhancer fromthe cytomegalo-virus immediate-early gene, the promoter, splice donor,and intron from the chicken β-actin gene, and the splice acceptor fromrabbit β-globin. The AAVrh10.CAG-hFXN-HA vector was produced asdescribed earlier (Rabinowitz, Rolling et al. 2002) in the Vector Coreat the University Hospital of Nantes(http://www.vectors.nantes.inserm.fr). The final titers of the twobatches used were 5.4×10¹² vg/ml and 2.15×10¹³ vg/ml, respectively.

Animal Procedures

Mice with a specific deletion of Fxn gene in cardiac and skeletal muscle(MCK-Cre-FxnL3/L-) (MCK mice) in 100% C57BL/6J background were generatedand genotyped as previously described (Puccio, Simon et al. 2001). Micewere maintained in a temperature and humidity controlled animalfacility, with a 12 hours light/dark cycle and free access to water anda standard rodent chow (D03, SAFE, Villemoisson-sur-Orge, France). Allanimal procedures and experiments were approved by the local ethicalcommittee for Animal Care and Use (Com'Eth 2011-07), and were performedin accordance with the Guide for the Care and Use of Laboratory Animals(National Institutes of Health). For biodistribution studies, threeweeks old wild-type mice were anesthetized by intraperitoneal injectionof ketamine/xylazine (75/10 mg/kg) to allow intravenous administrationby retro-orbital injection of AAVrh10.CAG-FXN at a dose of 5.10¹³ vg/kg,and sacrificed at 7 weeks of age (4 weeks post-injection). For genetherapy studies, three or seven weeks old MCK mice were anesthetized byintraperitoneal injection of ketamine/xylazine (75/10 mg/kg or 60/8mg/kg, respectively) to allow intravenous administration byretro-orbital injection of AAVrh10.CAG-FXN at a dose of 5.10¹³ vg/kg.Untreated MCK and WT mice littermates were injected with equivalentvolume of saline solution. Survival was evaluated daily and mice weightweekly. The mice cardiac function was evaluated under isofluoraneanesthesia (1-2%) by echocardiography by an experimenter blinded to micegenotype and treatment regimen, as previously described (Seznec, Simonet al. 2004). Animals were killed by CO₂ inhalation at 8, 15, 22 or 35weeks, and tissues samples for biochemical and molecular analysis wereimmediately frozen in liquid nitrogen. For histological analysis, micewere anesthetized by intraperitoneal injection of ketamine/xylazine andperfused with cooled saline solution. For histological analysis ofdorsal root ganglia, spinal cord and cardiac tissue was embedded in OCTTissue Tek (Sakura Finetechnical, Torrance, Calif.) and snap-frozen inisopentane chilled in liquid nitrogen. Samples of skeletal muscles weredirectly snap-frozen in isopentane chilled in liquid nitrogen. Forelectron microscopy analysis, small samples from the middle of leftventricle and its apex were collected, then fixed and embedded in Eponas previously described (Puccio, Simon et al. 2001).

Histopathology, Enzyme Histochemistry and Electron Microscopy

For histochemical analysis, 10 μm cryosections were stained either withhematoxylin and eosin (H&E), Sirius red and Fast green to labelextracellular collagen, or DAB enhanced Perls to label iron (Fe3+)deposits (Puccio, Simon et al. 2001).

Sirius red and fast green staining: Tissue sections were fixed with 10%paraformaldehyde in 0.1 M phosphate buffer (PBS), pH 7.4 for 10 min andthen incubated with a saturated solution of picric acid containing 0.1%Direct red 80 (Sigma) for 2 min, washed with 0.5% glacial acetic acidsolution followed by deionized water, and subsequently incubated in0.05% Fast Green solution for 5 min, and then washed with 0.5% glacialacetic acid solution. Finally, sections were dehydrated in gradedalcohols, cleared in Histosol Plus (Shandom) for 5 min and mounted usingPertex mounting medium (Histolab Products AB).

DAB-enhanced Perls iron staining: Tissue sections were fixed with 10%paraformaldehyde in 0.1 M phosphate buffer (PBS), pH 7.4 for 20 min andincubated in Perls solution (1% HCl, 1% Potassium Ferrocyanide) for 30min. Staining was enhanced by incubation in 0.025%3′-3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich), 0.005% H2O2in PBS buffer for 30 min, and then developed in the same buffer.Finally, sections were dehydrated in graded alcohols, cleared inHistosol Plus (Shandom) for 5 min and mounted using Pertex mountingmedium (Histolab Products AB).

Enzyme histochemical analyses: Succinate dehydrogenase (SDH) andCytochrome C Oxydase (COX) activities were performed on 10 μm cryostatsections of tissues, as previously described (Puccio, Simon et al.2001).

Electron microscopy analysis: Ultrathin sections (70 nm) of cardiactissue were contrasted with uranyl acetate and lead citrate and examinedwith a Morgagni 268D electron microscope, as described previously(Puccio, Simon et al. 2001).

Immunofluorescence and Image Acquisition

Cardiac and spinal cord tissue cryosections were fixed in 4% PFA for 10min, washed and then permeabilized in methanol at −20° C. for 20 min.Sections were blocked and permeabilized at the same time with PBS, 1%NGS, 5% BSA, 0.3% Triton X-100 for 1 h at room temperature (RT) and thenwashed in PBS, 0.2% Tween 1% BSA 1% NGS (PBS-TBN). Subsequently, tissueswere incubated overnight (O/N) at 4° C. with the rabbit polyclonalantibody against frataxin (FXN935)(1/250) diluted in PBS-TBN (Puccio,Simon et al. 2001). The Alexa fluor-594 goat anti-rabbit antibody(1/500) (Molecular Probes) was incubated for 2 h at RT. Sections werestained with Hoechst and mounted using Aqua-Polymount mounting medium(Polysciences, Inc.). For co-immunolabelling of HA-tag and prohibitin,the tissue section were washed in PBS, 0.05% Tween and then blocked O/Nat 4° C. in M.O.M.™ Mouse Ig Blocking Reagent (Vector Laboratories).Section were then incubated O/N at 4° C. with the mouse monoclonalantibody to HA tag (1/150) (Covence) diluted in M.O.M.™ diluent (VectorLaboratories). After washing, sections were incubated for 1 h at RT withthe goat anti-mouse antibody conjugated to Alexa Fluor-594 nm (1/500)(Molecular Probes) diluted in M.O.M.™ diluent. Subsequently, sectionswere washed and blocked in PBS, 0.3% Triton, 2% NGS for 1 h 30 at RT,washed and incubated for 2 h at RT with the rabbit polyclonal antibodyto prohibitin (1/150) (Abcam) diluted in PBS-BTN. The Alexa Fluor-488 nmgoat anti-rabbit antibody (1/500) (Molecular Probes) was incubated 1 h30 at RT with the goat anti-rabbit antibody conjugated to AlexaFluor-488 nm (Molecular Probes) diluted at 1/500 in PBS-BTN. Sectionswere stained with Hoechst and mounted using Aqua-Polymount mountingmedium (Polysciences, Inc).

Confocal analysis was performed on a Leica TCS SP2 upright confocalmicrosystem with a Plan Apo CS (numerical aperture 1.4) 63× objective.Observation of whole cardiac cryosections was performed on a Leica Z16APO A microsystem fitted with a QuanteM-S12SC camera and combined with a2× objective (39 mm working distance).

Quantitative Real-Time PCR Total

Total RNA was extracted from frozen heart pulverized with thePrecellys24 homogeniser (Bertin Technologies) and using TRI Reagent(MRC) according to the manufacturer's protocol and was treated withDNAse I treatment (Roche Biosciences). cDNA was generated by reversetranscription using the Transcriptor Fist strand cDNA synthesis kit(Roche biosciences). Quantitative RT-PCR was performed using the SYBRGreen I Master (Roche biosciences) and light Cycler 480 (Rochebiosciences) with primers described in Supplementary Table S3. 18Sribosomal RNA was used as internal standard.

Enzyme Activities

Tissues were immediately frozen in liquid nitrogen. The activities ofthe respiratory chain enzyme SDH (complex II), the citric acid cycleenzymes isocitrate dehydrogenase, and mitochondrial and cytosolicaconitases were determined as described (Puccio, Simon et al. 2001).

Immunoblot Analysis

Extracts of tissues were frozen in liquid nitrogen, and then homogenizedin lysis buffer containing Tris-HCl (280 mM, pH 6.8), 10% SDS, 50%glycerol. Total protein extract (10 μg or 50 μg) was analyzed onSDS-glycine polyacrylamide gels. Proteins were transferred tonitrocellulose membranes blocked with 5% non-fat milk and then incubatedwith the different primary antibodies, polyclonal anti-frataxin (R1250purified sera IGBMC, 1/1,000), anti-HA (Covance, 1/500),anti-mitochondrial aconitase (R2377 purified sera IGBMC, 1/20,000),anti-Ndufs3 (Invitrogen, 1/4,000), anti-SDH (Invitrogen, 1/4,000),anti-Rieske (Abcam, 1/5,000), anti-lipoic acid (Calbiochem, 1/5,000),anti-GAPDH (Millipore, 1/10,000) and monoclonal anti-beta-tubulin (2A2,IGBMC 1/1,000). Secondary antibody (goat anti-rabbit or anti-mouse IgG,respectively) coupled to peroxidase was diluted at 1/5,000 and used fordetection of the reaction with Supersignal Substrate Western blotting(Pierce), according to the manufacturer's instructions.

Statistical Analysis

All data are presented as mean±standard deviation of the mean (SD).Statistical analysis was carried out using Statview software (SASInstitute Inc). For statistical comparison of three experimental groups,one-way ANOVA followed by Scheffe's post-hoc test was used. A value ofP<0.05 was considered significant. For statistical comparison of twoexperimental groups, the bilateral Student's t-test was used. P<0.05 wasconsidered significant.

Quantitative PCR on Genomic DNA

Genomic DNA was extracted from heart by using a phenol-chloroformmethod. AAVrh10.CAG-FXN vector genome copy numbers were measured byquantitative PCR using the SYBR Green I Master (Roche Biosciences) andlight Cycler 480 (Roche Biosciences). The vector genome copy number percell (VGC) was evaluated as described (Piguet, Sondhi et al. 2012). Themouse genomic Adck3 sequence was used as internal control.

Results

Three week-old MCK mice that do not exhibit yet any clinical,echocardiographic nor biochemical signs of cardiac disease, received asingle intravenous injection of AAVrh10-CAG-hFXN at the dose of 5.4.1013vg/kg (n=9). Serial echocardiographic measurements identified that thetreatment efficiently prevented the development of the cardiac diseaseassociated with frataxin deficiency. While untreated MCK mice developeda rapidly progressing left ventricle hypertrophy associated with amassive geometric remodeling characterized by increased left-ventriculardiastolic diameter, the treated MCK mice were indistinguishable fromwild-type (WT) littermate animals (data not shown). In parallel,systolic function evaluated by the left-ventricular shortening fraction(SF) and the cardiac output gradually decreased in untreated mice, whilethe treated MCK mice showed no sign of altered ventricular contractility(data not shown). The absence of echocardiographic phenotype in thetreated MCK mice led to normal growth (data not shown) and survival (35weeks with no sign of disease), in contrast to untreated mice which dieat 65±10 days (FIG. 1A). To assess the cellular and molecular state ofthe cardiomyocytes, treated MCK mice were sacrificed at 35 weeks of agei.e. more than triple lifespan of untreated mice. Consistent with theevolution towards heart failure, the expression of atrial natriureticpeptide (ANP) and the brain natriuretic peptide (BNP), two markers ofpathology-induced stress program induced by hemodynamic overload wasmarkedly increased in the heart from untreated mice at 8 weeks comparedto WT (19 and 7 times, respectively, p<0.001) (FIG. 1B). In contrast, nodifference could be detected in the expression level of these twomarkers between the treated MCK mice and the WT littermates, supportingthe absence of pathology-induced stress programme due to hemodynamicoverload (FIG. 1B). Furthermore, while the expression of sarcoplasmicreticulum Ca2+ ATPase (Serca2a), a critical determinant of cardiacrelaxation responsible for diastolic Ca2+ reuptake from cytosol wasreduced in untreated mice (3.3 fold, p<0.01), treated MCK mice hadnormal Serca2a levels (FIG. 1C). Histological analysis confirmed apreserved overall heart organization in 35 week-old treated MCK mice,compared to the myocardial degeneration with cytoplasmic vacuolizationin the necrotic cardiomyocytes observed in untreated mice at 8 weeks ofage (data not shown). Furthermore, Sirius-red staining (data not shown)and collagen type I and III mRNA expression (data not shown) indicatedthe absence of myocardial post-necrotic fibrosis in treated animals, incomparison to the massive interstitial fibrosis present in untreated MCKmice at 8 weeks (data not shown).

Intravenous injection of AAVrh10-FXN led to robust viral transduction ofthe heart (20.85±6.3 vg/cell) and liver, but also of skeletal muscle anddorsal root ganglia (data not shown). Western blot analysis using ananti-FXN antibody, which equally detects human and mouse frataxins,demonstrated a significant overexpression (>10 fold) of AAVrh10-encodedfrataxin compared to endogenous frataxin of WT mice (data not shown).Sustained expression of the AAVrh10-encoded frataxin was seen over 35weeks (data not shown). Mitochondrial import and maturation of frataxinwas complete and non-saturated, as only the cleaved mature form of humanfrataxin was detected (data not shown). Immunohistochemistry analysisusing both anti-FXN and anti-HA antibodies showed a broad expression ofhuman frataxin throughout the heart of the AAV treated MCK mice, withclose to 100% of transduced cardiomyocytes in the LV, RV and septum,with some cardiomyocytes expressing higher levels (data not shown).Co-localization with prohibitin demonstrated the expected mitochondriallocalization of human frataxin (data not shown).

In line with the essential function of frataxin in regulating cellularFe—S cluster biogenesis, it is now commonly accepted that frataxindeficiency leads to a primary Fe—S cluster deficit followed by secondarymitochondrial iron accumulation. Indeed, while untreated MCK mice showeda strong deficit in the Fe—S mitochondrial aconitase (mAco) andsuccinate dehydrogenase (SDH) (41.3% and 79.8%, respectively) (data notshown), treated mice presented levels of activities similar to WTlittermates. Consistent with the widespread expression of hFXN in theheart after AAVrh10-CAG-hFXN injection, colorimetric staining of SDHactivity confirmed the correction of Fe—S biogenesis in over 95% ofcardiomyocytes (data not shown). While a substantial decrease in thelevels of all analysed mitochondrial Fe—S proteins, was detected inuntreated mice, as a result of the instability of the respective Fe—Sapo-proteins, treated mutants had levels equivalent to WT (data notshown). Similarly, expression of human frataxin prevented the decreasein activity of the Fe—S enzyme lipoic acid synthase, indirectlydemonstrated by normal levels of lipoic acid bound α-ketoglutaratedehydrogenase (KGDH) and pyruvate dehydrogenase (PDH) in treated animalsin comparison to untreated animals (data not shown). Consistent with theabsence of Fe—S cluster deficit, no cellular iron accumulation wasobserved in the cardiac tissue of treated mice (data not shown).Furthermore, we did not detect any sign of cellular iron homeostasisperturbation in treated animals (data not shown). Finally, electronmicroscopy analysis demonstrated a normal sarcomere organization of thecardiomyocytes and mitochondria ultrastructure in treated mice.Untreated animals showed sparse atrophied myofibrils and massivemitochondrial proliferation with abnormal collapsed or swollen cristaeand iron accumulation (data not shown). All together, these dataindicate that human frataxin gene transfer using AAVrh10 inpre-symptomatic MCK mice prevented the development of the mitochondrialFRDA cardiomyopathy at the molecular, cellular and physiological level.

While preventing the onset of the cardiomyopathy is an important step,at a clinical point of view it appears crucial to determine thetherapeutic potential of this gene therapy approach when cardiacdysfunction is already present. Mutant MCK mice were intravenouslyinjected with AAVrh10-CAG-hFXN at the dose of 5.4.1013 vg/kg (n=9) at 7weeks, when the ventricular remodeling and left ventricular systolicdysfunction are established, with a major decrease in cardiac output(60±9% versus control values), attesting of cardiac failure. One weekafter injection at 8 weeks of age, the LV function was alreadysignificantly improved, with a 49±5% ejection fraction and a decrease inLV hypertrophy and dilation in the treated mutant mice, whereasuntreated animals presented typical signs of heart failure (FIG. 2A).Echocardiographic parameters regarding cardiac function progressivelyimproved to reach WT values at 11-12 weeks of age, demonstrating acomplete recovery of the ventricular systolic function and anatomy. Thesurvival of the mice was significantly prolonged until at least 18 weeksof age (FIG. 2B). In accordance with the rapid reversion observed byechocardiography, human FXN was already strongly expressed one weekafter injection in heart of treated mutant mice and sustained over 22weeks, with a mitochondrial localization (data not shown). Similarly,the pathology-induced stress program induced by hemodynamic overload,reflected by the expression of ANP and BNP, was significantly decreasedone week after injection (8 weeks) in treated mice (FIG. 2C). By 22weeks, the expression level of ANP and BNP of treated MCK mice was closeto the expression level of WT animals, suggesting a normalization of thehemodynamic load. Furthermore, the expression of Serca2a progressivelyincreased in treated mice between 8 and 22 weeks, indicating thatdiastolic Ca2+ transport was likely restored (FIG. 2C). The reversal andcorrection of the cardiac phenotype correlated with a progressiveincreased in Fe—S proteins activities, mAco and SDH, in levels of theFe—S proteins Ndufs3, SDH, Rieske, as well as in the lipoic acid boundPDH and KGDH (FIG. 2D). At 22 weeks, some rare patches with low SDHactivity were detected in the cardiac tissue of treated mice (data notshown), corresponding to fibrotic scar probably already present at thetime of treated. Interestingly, collagen staining and expression (type Iand III) showed that interstitial cardiac fibrosis stopped one week postinjection (data not shown). Strikingly, a rapid correction of theultrastructure of the cardiac muscle was also observed one week afterinjection, with normal sarcomere organization and with a massivedecrease in mitochondria (data not shown). In correlation with a stillincomplete recovery of the biochemical phenotype one week aftertreatment, the mitochondria in the treated animals showed some signs ofpathology, with the presence of some swollen mitochondria presentingparallel stacks of cristae membranes (data not shown). However, by 22weeks, sarcomeres and mitochondria organizations completely recoveredwith no sign of pathological change. All together, these data indicatethat AAVrh10.CAG-hFXN treatment in symptomatic MCK mice resulted in arapid clinical, echocardiographic and biochemical improvement with acomplete correction of the FRDA cardiomyopathy.

CONCLUSION

Our data demonstrates that AAVrh10-mediated transfer of hFXN gene in themyocardium of a mouse model of severe FRDA cardiomyopathy not onlyprevents the onset of the disease for a sustained period, but also canreverse heart failure and cardiac remodelling. The correction isextremely rapid and efficient, with a striking reversal of themitochondrial abnormalities and biochemical Fe—S proteins deficit oneweek after treatment. Despite the severity of cardiac insufficiency atthe time of treatment, the cardiac recovery is rapidly progressive,reaching normality within 4-5 weeks of treatment.

Indeed, the correction of mitochondrial dysfunction in the mouse wasassociated with a progressive increase of sarcoplasmic reticulumCa2+-ATPase (Serca2a) gene expression involved in sarcoplasmic reticulumcalcium uptake from cytosol. Interestingly, a decrease in the expressionand activity of Serca2a has been identified in cardiomyocytes fromfailing human hearts. A rapid correction of the ultrastructure of thecardiac muscle was also observed and interstitial cardiac fibrosis wasstopped one week after treatment, preventing the dilation and massiveremodelling of the cardiac tissue. Fibrosis is an early manifestation ofFRDA cardiomyopathy and its importance in organ pathology anddysfunction is relevant to a wide variety of diseases, including heartdiseases.

In conclusion, delivery of a vector encoding hFXN in a mammalian modelof FRDA cardiomyopathy resulted in i) prevention of the development ofdisease symptoms in asymptomatic individuals and ii) reversal of diseasesymptoms in individuals who already exhibited cardiomyopathy,biochemical Fe—S cluster impairment, mitochondrial dysfunction andinterstitial cardiac fibrosis.

Thus, a gene that can reverse energy failure may be used for thetreatment and the prevention of a cardiomyopathy due to energy failure(like the use of FXN gene in the case of cardiomyopathy associated withFriedreich ataxia as explained in the examples).

REFERENCES

Throughout this application, various references, including United Statespatents and patent applications, describe the state of the art to whichthis invention pertains. The disclosures of these references are herebyincorporated by reference in entirety into the present disclosure.

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1. A method for treating cardiomyopathy in a Friedreich ataxia patientexhibiting cardiomyopathy, comprising administering to said patient intomyocardium of said patient a therapeutically effective amount of anAdeno-Associated Virus (AAV) vector which comprises a nucleic acidsequence encoding a frataxin (FXN) gene operably linked to regulatorysequences.
 2. (canceled)
 3. The method according to claim 1, whereinsaid FXN comprises the amino acids of SEQ ID NO:2.
 4. The methodaccording to claim 1 wherein the nucleic acid comprises the nucleic acidsequence of SEQ ID NO:1. 5-6. (canceled)
 7. The method according toclaim 1 wherein the AAV vector is an AAV1, AAV2, AAV3, AAV4, AA5, AAV6,AAV7, AAV8, AAV9, AAVrh10 vector.
 8. The method according to claim 7wherein the AAV vector is the AAVrh10 vector.
 9. The method according toclaim 1 wherein the AAV vector is administered by intracoronaryinjection.
 10. The method according to claim 1 wherein the AAV vector isadministered by intravenous injection.
 11. A pharmaceutical compositionfor treating cardiomyopathy in a Friedreich ataxia patient exhibitingcardiomyopathy, comprising an Adeno-Associated Virus (AAV) vector whichcomprises a nucleic acid sequence encoding frataxin (FXN) gene operablylinked to regulatory sequences, wherein said AAV vector is formulatedfor delivery into myocardium in said patient.
 12. (canceled)
 13. Thepharmaceutical composition according to claim 11 wherein the AAV vectoris an AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAVrh10vector.
 14. The pharmaceutical composition according to claim 13 whereinthe AAV vector is the AAVrh10 vector.
 15. The pharmaceutical compositionaccording to claim 11, wherein said FXN comprises the amino acids of SEQID NO:2.
 16. The pharmaceutical composition according to claim 11wherein the nucleic acid comprises the nucleic acid sequence of SEQ IDNO:1.
 17. The pharmaceutical composition according to claim 11 whereinthe AAV vector is formulated for intracoronary injection.
 18. Thepharmaceutical composition according to claim 11 wherein the AAV vectoris formulated for intravenous injection.
 19. The pharmaceuticalcomposition according to claim 11 wherein the AAV vector is formulatedfor epicardiac injection.
 20. The pharmaceutical composition accordingto claim 11 wherein the AAV vector is formulated for endomyocardicinjection.
 21. The pharmaceutical composition according to claim 11,wherein said FXN comprises amino acids 81-210 of SEQ ID NO:2.
 22. Thepharmaceutical composition according to claim 11 wherein the nucleicacid comprises a variant of the nucleic acid of SEQ ID NO: 1 whichencodes amino acids 81-210 of SEQ ID NO:
 2. 23. The method according toclaim 1 wherein said FXN comprises amino acids 81-210 of SEQ ID NO:2.24. The method according to claim 1 wherein the nucleic acid includes avariant of the nucleic acid sequence of SEQ ID NO: 1 which encodes aminoacids 81-210 of SEQ ID NO:
 2. 25. The method according to claim 1wherein the AAV vector is administered by endomyocardic injection. 26.The method according to claim 1 wherein the AAV vector is administeredby epicardiac injection.